Zhang Jing - "Effect of chronic fluorosis on calcium signal pathway in rat brain hippocampus" Master's Thesis (2010)
http://cdmd.cnki.com.cn/Article/CDMD-10 ... 241383.htm
【Summary】: Appropriate amount of fluoride is the basis for maintaining the normal physiological activities of the body, but fluorine is a cumulative protoplasmic poison. Long-term excessive intake of fluoride can cause damage to bone organs such as skeletal fluorosis, dental fluorosis, and non-skeletal organs such as blood and nervous system. Damage. Nerve tissue is highly sensitive to fluorine. Hippocampus is one of the target sites of fluorosis in the central nervous system, and its nerve cells are susceptible to plastic changes due to fluorine. In recent years, through the research on the neurotoxicity mechanism of fluoride, people have mainly proposed the G protein signal transduction theory and the oxidative stress theory. At present, it is generally believed that the damage of the nervous system caused by neurotoxins is closely related to the imbalance of Ca2+ homeostasis in the G protein signal transduction pathway. The mechanism may be related to the expression of nuclear factor kappa-B (NF-κB) in nerve cells that are sensitive to oxidative stress and regulated by calcium ions. Therefore, this experiment replicated the drinking water-type chronic fluorosis animal model, based on the observation and detection of dental fluorosis caused by fluorosis, abnormal blood fluoride levels, and changes in the morphological structure of hippocampal nerve cells. For the first time, calcium ions were used in the neurotoxicity study of fluoride. The signal pathway is combined with the oxidative stress theory to detect the effect of fluorosis on the Ca2+ concentration in rat hippocampal synapses and the expression of CaMKⅡ, c-fos, NF-κB, Bcl-2 and Bax protein in the calcium signal pathway.
Research method: 152 first weaned male SD rats were randomly divided into four groups, namely the control group (drinking tap water, water fluoride content is less than 0.5mg/L), low fluoride group, medium fluoride group, high fluoride group (drinking water containing Sodium fluoride is 15mg/L, 30mg/L, 60mg/L). Each group used the prepared solution as the only source of drinking water, and they were free to eat and drink. The feeding time was 18 months. At the initial stage of fluoride exposure (3 months old), the rats were tested for dental fluorosis and blood fluoride, and the rats were decapitated in two batches at the middle of fluoride exposure (9 months old) and at the end of fluoride exposure (18 months old), and the hippocampus was detected by HE staining. Pathological changes in CA3 area, fluorescent indicator Fura-2/AM to detect the changes of Ca2+ concentration in hippocampal synapses in rats with chronic fluorosis, and immunohistochemical method to detect CaMKⅡα, c-fos, NF-κBρ65, Bcl-2 in hippocampal CA3 area And the change of Bax protein expression. 1. Excessive fluoride intake will cause dental fluorosis in rats. With the increase of fluoride concentration, the symptoms of dental fluorosis in rats will increase, and the incisors will be obviously deformed, defective and chalky horizontal stripes; the blood fluoride concentration of each fluoride group is Compared with the control group, significantly or extremely significantly increased (P0.05 or P0. 01). It shows that the animal fluorosis model has been successfully replicated. 2. HE staining results show that chronic fluorosis can damage the morphological structure of rat nerve cells. Compared with the control group, the hippocampal nerve cells in the fluoride group were sparsely distributed, the cell outline disappeared, and vacuoles appeared. And this change becomes more significant with the increase of fluorine exposure time and fluorine concentration. 3. The results of the detection of the concentration of synaptosome Ca2+ show that with the increase of months of age in rats, the concentration of synaptosome Ca2+ in the control group has a tendency to increase, and fluoride treatment aggravates the overload of Ca2+. Mid-stage fluoride exposure: Compared with the control group, the Ca2+ concentration of synaptosomes in the medium fluoride group and the high fluoride group increased significantly or extremely significantly (P0.05 or P0.01); the end of fluoride exposure: compared with the control group, medium fluoride The Ca2+ concentration of synaptosomes in the high fluoride group and the high fluoride group increased significantly (P0.01). 4. IHC results show that with the increase of months of age in rats, the protein expressions of CaMKⅡα, c-fos, and NF-κBρ65 in the control group and each fluorine-exposed group all increased at different levels, and the increase in fluoride exposure time and fluoride dose increased this This change is more significant. Mid-stage fluoride exposure: Compared with the control group, the expression of CaMKⅡα in the medium and high fluoride groups was significantly increased (P0.05), the expression of c-fos was significantly or extremely significantly increased (P0.05 or P0.01), NF- Although there is no significant difference in the expression of κBρ65, its expression tends to rise with the increase of fluoride dose; the end of fluoride exposure: Compared with the control group, the expression of CaMKⅡαc-fos in the medium fluoride group and the high fluoride group increased significantly (P0. 01), the expression of NF-κBρ65 in the fluorine group was significantly increased (P0.05). 5. IHC results show that with the increase of the age of the rats, the expression levels of Bcl-2 and Bax proteins in the control group and each fluorine-exposing group have different levels of decline, and the increase in the fluorine exposure time and the fluoride dose makes this change more Is significant. Mid-stage fluoride exposure: Compared with the control group, the expression of Bcl-2 in the medium and high fluoride groups decreased significantly or significantly (P0.05 or P0.01), and the expression of Bax decreased significantly (P0.01); end of fluoride exposure ：Compared with the control group, the expression of Bcl-2 in the middle fluoride group and the high fluoride group was significantly or very significantly decreased (P0.05 or P0.01), and the expression of Bax in the low fluoride group, the middle fluoride group and the high fluoride group was significantly or extremely significant Decrease (P0.05, P0.01, P0.01). At the same time, the middle period of fluoride exposure: the ratio of Bax to Bcl-2 (Bax/Bcl-2) tends to increase with the time and concentration of fluoride exposure; the end of fluoride exposure: Compared with the control group, the middle fluorine group has a higher The ratio of Bax/Bcl-2 in the fluoride group increased significantly (P0. 05), suggesting that nerve cells are in apoptotic state. In summary, chronic fluoride exposure has toxic effects on the central nervous system. Fluoride can cause changes in the morphology and structure of hippocampal nerve cells, causing hippocampal synaptosome Ca2+ overload, which in turn leads to abnormal protein expression of CaMKⅡα, c-fos, NF-κBρ65, Bcl-2 and Bax in the calcium ion signaling pathway, and finally induces nerve cells Prone to apoptosis. In the process of aging, the degenerative changes of nerve cells can also lead to apoptosis of rat nerve cells, and fluoride treatment exacerbates this change. This study further clarified the molecular mechanism of the effect of fluoride neurotoxicity on the calcium ion signaling pathway, and provided a theoretical basis for in-depth exploration of the molecular biological mechanism of chronic fluorosis to the central nervous system. In addition, the expression of NF-κB has a sensitive dose-effect relationship with the time and concentration of fluoride exposure, and may be a target molecule related to fluoroneurotoxicity. This provides a certain theoretical basis for the development of biomarkers of fluoroneurotoxicity.
《浙江师范大学》 2010年收藏 | 手机打开
【摘要】： 适量氟是维持机体正常生理活动的基础,但氟是一种蓄积性原生质毒物,机体长期过量摄入氟可引起氟骨症、氟斑牙等骨相器官的损害以及血液、神经系统等非骨相器官的损害。神经组织对氟的敏感性很高,海马作为中枢神经系统中氟中毒的靶部位之一,其神经细胞易受氟的影响而发生可塑性变化。近年来,通过对氟神经毒性作用机制的研究,人们主要提出了G蛋白信号转导理论与氧化应激理论。 目前普遍认为,神经毒物引起的神经系统损伤与G蛋白信号转导通路中Ca2+稳态失调有密切关系。其机制可能与神经细胞内对氧化应激敏感且受钙离子调控的核转录因子(nuclear factor kappa-B, NF-κB)表达等有关。因此,本实验通过复制饮水型慢性氟中毒动物模型,以观察和检测氟中毒引起氟斑牙、血氟水平异常及海马神经细胞形态结构改变为基础,首次在氟的神经毒性研究中将钙离子信号通路与氧化应激理论结合,检测氟中毒对大鼠海马突触体内Ca2+浓度以及钙离子信号通路中CaMKⅡ、c-fos、NF-κB、Bcl-2及Bax蛋白表达的影响。 研究方法：152只初断乳雄性SD大鼠随机分为四组,即对照组(饮用自来水,水氟含量低于0.5mg/L),低氟组、中氟组、高氟组(饮水含氟化钠分别为15mg/L、30mg/L、60mg/L)。各组以配制的溶液作为饮水唯一来源,自由摄食、饮水,饲养时间为18个月。染氟初期(3月龄)对大鼠进行氟斑牙和血氟检测,染氟中期(9月龄)和染氟结束(18月龄)分两批断头处死大鼠,HE染色检测海马CA3区病理学改变,荧光指示剂Fura-2/AM检测慢性氟中毒大鼠海马突触体内Ca2+浓度的变化,免疫组化法检测海马CA3区CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax蛋白表达的改变。 1、过量氟摄入会导致大鼠氟斑牙发生,随染氟浓度升高,大鼠氟斑牙症状加剧,切齿明显变形、缺损并出现白垩状横纹；各染氟组血氟浓度与对照组相比显著或极显著升高(P0.05或P0.01)。表明动物氟中毒模型复制成功。 2、HE染色结果显示,慢性氟中毒可损伤大鼠神经细胞形态结构。与对照组相比,染氟组大鼠海马神经细胞分布稀疏,细胞轮廓消失,并出现空泡。且这种变化随染氟时间和染氟浓度的递增而更为显著。 3、突触体Ca2+浓度检测结果显示,随大鼠月龄增长,对照组突触体Ca2+浓度有升高的趋势,而染氟处理加剧了Ca2+的超载。染氟中期：与对照组相比,中氟组、高氟组突触体Ca2+浓度显著或极显著升高(P0.05或P0.01)；染氟结束：与对照组相比,中氟组、高氟组突触体Ca2+浓度极显著升高(P0.01)。 4、IHC结果显示,随大鼠月龄增长,对照组及各染氟组CaMKⅡα、c-fos、NF-κBρ65蛋白表达均有不同水平的上升,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期：与对照组相比,中氟组、高氟组CaMKⅡα表达显著升高(P0.05),c-fos表达显著或极显著升高(P0.05或P0.01),NF-κBρ65表达虽无显著性差异,但随染氟剂量升高其表达有上升的趋势；染氟结束：与对照组相比,中氟组、高氟组CaMKⅡαc-fos表达极显著升高(P0.01),中氟组NF-κBρ65表达显著升高(P0.05)。 5、IHC结果显示,随大鼠月龄增长,对照组及各染氟组Bcl-2和Bax蛋白表达水平均有不同水平的下降,且染氟时间和染氟剂量的递增使这种变化更为显著。染氟中期：与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P0.05或P0.01),Bax表达极显著下降(P0.01)；染氟结束：与对照组相比,中氟组、高氟组Bcl-2表达显著或极显著下降(P0.05或P0.01),低氟组、中氟组、高氟组Bax表达显著或极显著下降(P0.05,P0.01,P0.01)。同时,染氟中期：Bax与Bcl-2比值(Bax/Bcl-2)随染氟时间和染氟浓度的递增有升高的趋势；染氟结束：与对照组相比,中氟组、高氟组Bax/Bcl-2比值显著上升(P0.05),提示神经细胞处于凋亡状态。 综合上述,慢性氟暴露对中枢神经系统有毒性作用。氟可导致海马神经细胞形态结构发生改变,引起海马突触体Ca2+超载,继而导致钙离子信号通路中CaMKⅡα、c-fos、NF-κBρ65、Bcl-2及Bax的蛋白表达异常,最终诱导神经细胞趋于凋亡。衰老过程中,神经细胞的退行性改变也可导致大鼠神经细胞凋亡,而染氟处理加剧了这种变化。本研究进一步阐明了氟神经毒性对钙离子信号通路影响的分子机理,为深入探讨慢性氟中毒对中枢神经系统毒性作用的分子生物学机制提供了一定的理论基础。此外,NF-κB的表达与染氟时间、浓度有敏感的量效关系,可能是氟神经毒性相关靶分子,这为开展氟神经毒性的生物标志物研究提供一定的理论依据。
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